Thiazolyl peptide antibiotics, including thiostrepton, the thiocillins and the GE2270 family, disrupt bacterial protein synthesis selectively with nanomolar affinity either for a site on the 50S ribosomal subunit or on elongation factor EF-TU. We and others have recently shown that the pyridine/dihydropyridine ring-containing macrocyclic scaffold necessary for the antibiotic activity of this natural product class arises from posttranslational modification of nascent ribosomally generated preproteins. Thus, the C-terminal 14 residues of the prethiocillin 52mer undergo a cascade of 14 posttranslational modifications, including 6 Cys to thiazoles, two threonines to dehydrobutyrines and two serines to the central pyridine heterocycle. In the thiocillin producer B, subtilis ATCC 14579 we have shown that preprotein gene replacement will allow in vivo analysis of requirements for the posttranslational cascade. This proposal will investigate the timing, order, and mechanisms of the posttranslational cascade modifications with the ultimate aim of reprogramming the machinery to understand target specificity (50S ribosomal subunit vs EF-Tu) and to optimize antibiotic activities. This will include study of the 26-atom macrocyclic thiocillin and also cloning, expression, and evaluation of the gene clusters encoding the 29 atom macrocyclic GE 37468 (which targets EF-Tu not the 50S ribosomal subunit) and the 35 atom macrocyclic berninamycin A, in which the 7 C-terminal serines are processed posttranslationally, five of them to dehydroAla, one to an oxazole, and the other condensed to form the embedded pyridine: we wish to understand how the distinct posttranslational fates of these serines during antibiotic scaffold maturation are controlled.